Page 41 - วารสารกรมการแพทย์แผนไทยฯ ปีที่ 18 ฉบับที่ 2

J Thai Trad Alt Med Vol. 18 No. 2 May-Aug 2020 263

radical solution was adjusted with absolute [100 mM Tris–HCl (pH 7.5), 100 mM imidazole,
ethanol to an absorbance of 0.7 ± 0.02 at 732 15 mM MgCl and 1.0 mg/mL BSA], 25 ml of
2
nm. Ten microliters of T. triquetrum extracts 10 mM EGTA, 25 ml of PDE solution and 25

were mixed with 190 mL of ABTS radical ml of test sample or only solvent (5% DMSO)
solution in 96-well plates. The decrease of as a control. The reaction mixture was mixed

absorbance was measured at 732 nm using a with a substrate, 25 ml of 1 mM [3H] cGMP and
microplate reader. The standard curve was lin- incubated at 30 ˚C for 10 min. After that, the
ear between 0.025 and 0.9 mM Trolox. Results reaction was stopped by placing the tube in

were expressed in the concentration of Trolox boiling water for 1 min and cooled for 5 min. For
per g of T. triquetrum extract. the second enzymatic reaction, 25 ml of 2.5 mg/

5. Phosphodiesterase-5 inhibitory activity mL snake venom containing 5′-nucleotidase
Erectile dysfunction (ED) is one of sexual enzyme was added to the reaction mixture,
dysfunction that correlates with phospho- incubated at 30 ˚C for 5 min. Then, 250 ml of

diasterase-5 activity. Phosphodiesterase-5 20 mM Tris–HCl, pH 6.8 (buffer 1) was added.
(PDE-5) Inhibitors have been used as first-line The reaction mixture was transferred to a
therapy for ED. The mechanism of action of diethylaminoethyl (DEAE) ion exchange resin

this drug class is inhibition of the enzyme column and eluted 4 times with 500 ml of buffer
PDE-5 which is an enzyme breaking down 1 to obtain the hydrolysis product, uncharged
cGMP. Vasodilatory effect enhanced by cGMP [3H] guanosine. The eluant was mixed with a

improving penile erection. The basic screen- scintillant cocktail and the radioactivity was
ing of promising agent for enhancing sexual measured using a β-counter. The PDE in the

performance is often measured by phosphodia- study was standardized to have a hydrolysis
sterase-5 (PDE-5) inhibitory activity. Current activity of 15–20% of the total substrate counts.
[13]
study, phosphodiasterase-5 (PDE-5) inhibitory The PDE inhibitory activity is calculated from

activity of T. triquetrum extracts was investi- Eq. (1). The calculation of hydrolysis is shown
gated by Herb tech, Faculty of Pharmaceutical in Eq. (2).

Sciences, Naresuan University, Thailand. The
method was described in brief ; .. (1)
[14]
PDE-5 was obtained from mouse lung

tissue. It was performed following as Tem- where % hydrolysis sample and % hydrolysis
kifthawon and colleagues report. The reac- control were the enzyme activities of the
[5]
tion mixture was composed of 25 ml of buffer A sample and solvent (1% DMSO) used in the